BACKGROUND:Chordoma is a midline neoplasm accounting for approximately 20% of primary spinal tumors. Due to chordoma's locally aggressive nature, patients experience high rates of disease progression and have limited treatment options, highlighting an unmet clinical need. This underscores the importance of exploring novel therapeutic strategies. Oncolytic viral (OV) therapy uses genetically modified viruses to selectively replicate in tumor cells, mediate tumor cell lysis, and initiate a pro-inflammatory response within the infected tumor microenvironment (TME). The aim of the study was to evaluate the anti-chordoma effect of the replicating oncolytic adenovirus Delta-24-RGD. METHODS:The efficacy of Delta-24-RGD was assessed using in vitro approaches, ex vivo bone scaffolds, and in vivo murine models. Additionally, we explored the underlying mechanism of OV cytotoxicity, immunogenic cell death (ICD), brachyury modulation, and reshaping of the TME towards a pro-inflammatory state. RESULTS:Delta-24-RGD achieved viral infectivity, oncolysis, and ICD across multiple human chordoma cell lines and ex vivo bone scaffold models. In vivo murine xenograft models of human CH22 and U-CH1 chordoma treated with Delta-24-RGD resulted in tumor volume reduction, enhanced overall survival, and microenvironmental transcriptional modulation. Analyzing a human chordoma tissue microarray, the immunosuppressive macrophage marker CD163 was associated with shortened recurrence-free survival. Using macrophage/chordoma co-culture, OV infection achieved a reduction in macrophage CD163 expression and a concomitant pro-inflammatory macrophage polarization. CONCLUSIONS:The immunosuppressive chordoma TME is associated with clinical outcomes and our data suggests OV infection reverses this deleterious immunosuppressive profile. These studies provide a framework for future clinical implementation among chordoma patients.

Abstract Amongst solid cancers, melanoma brain metastases (MBM) have the highest likelihood of metastasizing to the brain. The use of immune checkpoint inhibitors (ICI) has emerged as first line therapy; however, intracranial progression and acquired resistance highlight the need for novel immunotherapeutic strategies for this patient population. Oncolytic viral (OV) therapy leverages tumor cell replication machinery to selectively replicate and kill tumor cells while promoting anti-tumor immunity. The oncolytic adenovirus Delta-24-RGDOX (RGDOX) has been shown to function as a TME modulator in both preclinical and clinical settings. Here, we investigated the anti-tumor efficacy of RGDOX combined with ICIs in synchronous extracranial—intracranial (s.c.-i.c.) and intracranial—intracranial (i.c.-i.c.) murine melanoma models (B16F10 and D4M). Using this approach, we inoculated the s.c. (s.c.-i.c. model) or i.c. (i.c.-i.c.) tumor with RGDOX and monitored for viral induced cytotoxicity within the infected tumor while assessing for the simultaneous generation of intracranial antitumor immunity against the uninfected tumor. The combinatorial strategy of RGDOX with PD-1/CTLA-4 inhibition significantly reduced tumor growth within the infected and distant intracranial tumor while also improving survival in tumor bearing mice. Through a series of immune depletion experiments, therapeutic efficacy was determined to be mediated through CD8+ T cells. Additionally, eradication of subcutaneous tumor led to immunologic memory preventing intracranial tumor formation. Immunophenotyping and spatial profiling demonstrated that treatment boosted anti-tumor immunity in both infected and uninfected intracranial tumors through enhanced T-cell activity, and increased infiltration of both dendritic cells and inflammatory macrophages. In contrast, immunosuppressive populations were reduced in both infected tumors and uninfected intracranial tumors following treatment. These findings provide the foundation for our trial—Dose Ranging, Toxicity Seeking, Phase 1 Trial of Oncolytic Adenoviral Therapy for Melanoma Intracranial and Extracranial Metastases (NCT07444606)—and suggest oncolytic viral therapy is a promising therapeutic approach for the generation of antitumor immunity against MBM.

We identified an important oncogenic role for protocadherin 7 (PCDH7), a cell surface protein frequently overexpressed in lung adenocarcinoma and associated with poor clinical outcome. Pcdh7 depletion reduces tumor burden and prolongs survival in KrasLSL-G12D; Tp53fl/fl mice. These findings nominate this cell surface protein as an actionable therapeutic target and highlight the therapeutic potential of PCDH7 inhibition for non-small cell lung cancer. We report the development and characterization of high-affinity anti-PCDH7 monoclonal antibodies (mAbs) that inhibit downstream mitogen-activated protein kinase (MAPK) pathway activation and suppress tumor growth in multiple mutant KRAS-driven models. A lead mAb (mAb7) sensitized tumors to the US Food and Drug Administration-approved MAPK kinase inhibitor trametinib and the KRASG12C inhibitor adagrasib. A humanized mAb7-IgG1 (Hu-mAb7) exhibited antibody-dependent cellular cytotoxicity and Fc-mediated immune effector killing of tumor cells in vivo. Moreover, a murinized antibody (Ms-mAb7) improved antitumor immunity in a KrasG12D syngeneic tumor model by enhancing infiltration and activation of cytotoxic immune cells. These findings provide an important advance in the clinical development of PCDH7-targeting antibodies for lung cancer treatment.

2048 Background: Oncolytic viruses have shown promise in clinical trials for solid tumors, including glioma and melanoma, but only a subset of patients benefits. We previously showed that arming the oncolytic adenovirus Delta-24-RGD with OX40L can enhance antitumor immunity. To further boost efficacy, we developed Delta-24-RGDOX-IL15, co-expressing OX40L and IL-15, and tested it in preclinical models of primary and metastatic brain tumors. Methods: To evaluate IL-15 and its receptor (IL15RA) expression in patients with glioma and melanoma, we conducted gene expression and survival analysis using the GEPIA web server, integrating RNA sequencing data from TCGA and GTEx. Transgene expression in Delta-24-RGDOX-IL15 was assessed via flow cytometry and ELISA, while viral potency was evaluated using replication and cell viability assays. Anti-tumor activity was tested in syngeneic intracranial models derived from mouse diffuse midline (DMG) glioma and melanoma cell lines in C57BL/6 mice, both of which expressed GD2 and luciferase. Tumor growth was monitored with bioluminescent imaging, survival with Kaplan-Meier analysis, and immune profiling of the tumor microenvironment using flow cytometry. Results: GEPIA analysis showed that melanoma had higher expression of IL-15 and IL-15RA compared to glioma. In melanoma patients, higher expression of IL-15RA or IL-15 was linked to better overall survival (P < 0.005), while no survival difference was found in patients with glioma. Delta-24-RGDOX-IL15 infected and co-expressed OX40L and IL-15 effectively in mouse glioma and melanoma cells, and induced potent oncolysis. Delta-24-RGDOX-IL15-infected tumor cells significantly enhanced the oncolysis activity of GD2 CAR T cells in culture. Intratumoral injection of the virus also resulted in better tumor reduction and improved survival in C57BL6 mice with gliomas derived from mouse DMG cells while no significant toxicity was observed. Additionally, locoregional therapy with Delta-24-RGDOX-IL15 induced a systemic inflammatory response in the tumor microenvironment, characterized by increased frequency of T cells and reduced that of myeloid cells. Conclusions: Higher expression of IL-15/IL-15RA is associated with better survival in patients with melanoma. Delta-24-RGDOX-IL15 demonstrates potent oncolytic activity in both glioma and melanoma cell lines. In the intracranial brain tumor mouse model, it exhibited promising anti-tumor effects with enhanced T cell stimulation and minimal toxicity. Delta-24-RGDOX-IL15 is a promising candidate for combination with cellular therapies in both primary and metastatic brain tumors.

Advanced solid tumors pose significant clinical challenges due to a heterogeneous and immunosuppressive tumor microenvironment, which limits the efficacy of approved therapies. Based on promising clinical trial results with the oncolytic adenovirus (OA) Delta-24-RGD in glioma patients, we previously demonstrated that Delta-24-RGDOX, engineered to express the immune co-stimulatory molecule OX40 ligand (OX40L), enhances local and systemic anti-tumor immunity via an in situ autovaccination effect in immunocompetent mice bearing orthotopic intracranial (i.c.) gliomas or disseminated subcutaneous (s.c.)/i.c. melanomas. To further enhance therapeutic efficacy, we developed Delta-24-RGDOX-IL15, a novel OA co-expressing IL-15 and OX40L. IL-15 is known to activate NK and T cells and support the persistence of CD8⁺ memory T cells. Delta-24-RGDOX-IL15 maintained robust viral replication and oncolytic activity comparable to its predecessors and more effectively stimulated T cell responses, including CAR T cells, against cancer cells expressing corresponding tumor-associated antigen. In C57BL/6 mice bearing syngeneic GD2-expressing tumors, including i.c. gliomas or disseminated s.c./i.c. melanomas, intratumoral delivery of Delta-24-RGDOX-IL15 into the i.c. glioma or s.c. melanoma significantly reduced microglia and myeloid cell frequency while increasing T cell frequency and activation of NK, dendritic, and T cells within the tumor-bearing brain hemisphere from the mice of the two mouse models. Notably, when combined with murine GD2 CAR T cells, Delta-24-RGDOX-IL15 enhanced CAR T cell accumulation in both treated and distant, untreated brain tumors, leading to long-term survival in 20% of mice with s.c./i.c. melanomas. Currently, we are optimizing the treatment regimen to achieve better efficacy in both mouse models. Together, these findings demonstrate the potent immunostimulatory and therapeutic activity of Delta-24-RGDOX-IL15 and its potential to synergize with CAR T cell therapy in treating advanced primary or metastatic brain tumors.

GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. Despite advances in immunotherapy, current treatment modalities have only provided a modest benefit to GBM patients. We propose that enhancing the efficacy of immunotherapy for GBM requires approaches that increase T cell infiltration and reprogram the myeloid cells to a more pro-inflammatory state. We previously reported that S100A4 is a critical regulator of immune suppressive myeloid and T cell phenotypes and also a major regulator of glioma stem cells in GBM. Our preliminary data show that targeting S100A4 in either glioma cells or stromal cells is sufficient to reprogram the tumor immune landscape and extend survival of glioma bearing mice, indicating that S100A4 is a promising therapeutic target. We recently developed a S100A4 blocking monoclonal antibody; however, this antibody does not cross the blood brain barrier (BBB) efficiently to confer significant therapeutic benefit in glioma bearing mice. To overcome this challenge, we generated a bispecific S100A4-TfR antibody (BsA) that allows robust BBB penetration and accumulation in the brain. We assessed efficacy of BsA treatment in a highly aggressive and therapy-resistant, syngeneic mouse model of GBM (SVP). Using ELISA, confocal immunofluorescent microscopy, flow cytometry, and bulk RNA sequencing, we report that BsA treatment accumulates in the gliomas and reprograms the tumor immune microenvironment to increase T cell infiltration and pro-inflammatory activation. Furthermore, BsA treatment in combination with αPD1 checkpoint blockade significantly extends survival in the SVP mouse glioma model. Combination treated tumors exhibit extensive CD8 T cell infiltration and increased tumor reactive CD4 and CD8 T cells compared to controls. In addition, the BsA is taken up intracellularly within GBM cells and depletes S100A4 and nuclear SOX2 protein. In vitro treatment of GBM stem cell cultures with the BsA reduced self-renewal, as measured by the secondary sphere formation assay. These results indicate that the BsA is a promising agent to reprogram the GBM immune landscape and target GBM cell stemness through inhibition of both extracellular and intracellular S100A4 protein. We are currently performing in vivo efficacy studies in additional murine glioma models, and deeper mechanistic studies using single cell RNA sequencing and functional assays. Thomas Wong, Jia-Shiun Leu, Xuejun Fan, Fransisca Leonard, Maryam Faisal, Reece Kang, Yaqoob Ali, Nourhan Abdelfattah, Ningyan Zhang, Kyuson Yun. A bispecific S100A4/TFR antibody to reprogram the GBM tumor microenvironment and target GBM stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6069.

Malignant gliomas pose a significant therapeutic challenge. Remarkably, complete tumor regressions have been observed in a subset of patients with recurrent malignant gliomas treated with Delta-24-RGD oncolytic adenovirus in phase 1 and 2 clinical trials. Radiographical pseudo-progressions preceded tumor regressions, suggesting the involvement of an anti-tumor immune response. To evaluate the role of T cells, we confirmed CD3+ T cell infiltration in tumors obtained during a phase 1 clinical trial (NCT00805376) and further investigated the T cell landscape using T cell receptor (TCR) sequencing. Notably, tumors resected two weeks post-treatment (n=9) exhibited significantly reduced TCR diversity compared to pre-treatment tumors (n=6, p = 0.036). An independent dataset of peripheral blood from pediatric DIPG patients treated with Delta-24-RGD (NCT03178032) confirmed the decrease in TCR diversity post-treatment (n=10, p = 0.043). Notably, patients who retained higher TCR diversity post-treatment demonstrated prolonged survival (p = 0.022), strongly suggesting a link between immune repertoire retention and clinical outcomes. Further analysis of pre- and post-treatment samples revealed limited overlap in T cell clonotypes, with only 20% of the post-treatment clonotypes detected in pre-treatment tumors. The degree of overlap correlated with patient survival (p = 0.039). Using GLIPH2 clustering, we identified 617 specificity groups among 2,737 tumor-derived CDR3b sequences, with CDR3 motifs including RHAG, KSFS and S%SPKET shared with an independent glioma dataset, potentially indicating glioma-reactive T cells. In summary, our findings provide for the first time a clinically relevant assessment of T cell dynamics in oncolytic-adenovirus-treated glioma patients. Based on our clinical data revealing the significance of TCR diversity and repertoire overlap, we demonstrate that immune profiling is a potential biomarker for therapy response and survival, paving the way for more precise and effective immunotherapy strategies for glioma.

There is no effective treatment for H3K27-altered diffuse midline glioma (DMG). The hallmark mutation, H3K27M, observed in approximately 80% of the patients, influences universal H3K27 hypomethylation and subsequent hyperacetylation at this residue and is associated with tumor aggressiveness. We and our collaborators have demonstrated the therapeutic efficacy of the oncolytic adenovirus Delta-24-RGD in phase I clinical trials (NCT00805376 and NCT03178032) for the treatment of both adult and pediatric patients with gliomas. Based on previous studies outlining the process of early adenoviral protein E1A binding to the acetyltransferase enzymes P300/CBP during virus infection and the documented cases of P300-mediated tumor immunosuppression in several cancer types, we hypothesized that pharmacological inhibition of P300 would enhance the antitumor immunity mounted by Delta-24-RGD in DMG and improve survival. Histone post-translational modification analyzed by mass spectrometry demonstrated the widespread hypoacetylation effect of Delta-24-RGD. Validating these findings with western blot studies, we demonstrated that Delta-24-RGD induced loss of H3K27ac and H3K18ac in a panel of clinically significant human and murine DMG cells. Furthermore, we observed synergistic anti-glioma effects of Delta-24-RGD and P300/CBP acetyltransferase inhibitors, C646 and CPI-1612, in vitro (ZIP scores ranging up to 15.90). Proteome array experiments revealed an increase in expression of inflammatory cytokines involved in T-cell recruitment and activation, 14 days post-virus treatment in the combination-treated mice, indicating that P300 inhibition maintains an inflammatory milieu after Delta-24-RGD. Importantly, the combination of Delta-24-RGD and P300 inhibition reduced tumor burden in clinically relevant models of pediatric DMG. In conclusion, our results demonstrate that the dual target of the oncohistone driver of DMG induces a potent anti-cancer effect, providing a strong rationale for the translation of this combination to the clinical scenario.

Chordomas are locally aggressive neoplasms accounting for approximately 20% of primary spinal tumors. Patients experience high rates of disease progression and limited treatment options, highlighting an unmet clinical need and the importance of identifying novel therapeutic strategies for chordomas. Oncolytic viral (OV) therapy uses genetically modified viruses which selectively replicate in tumor cells, mediate tumor cell lysis, and initiate a pro-inflammatory response within the infected tumor microenvironment (TME). This study evaluates the anti-chordoma effect of the oncolytic adenovirus Delta-24-RGD. The efficacy of Delta-24-RGD was assessed using in vitro approaches, ex vivo bone scaffolds, and in vivomurine models. Additionally, we explored the underlying mechanism of OV cytotoxicity, immunogenic cell death (ICD), brachyury modulation, and reshaping of the TME towards a pro-inflammatory state. Delta-24-RGD achieved viral infectivity, oncolysis and ICD across multiple human chordoma cell lines and ex vivo bone scaffold models. Viral infection was associated with concomitant brachyury downregulation within both infected and uninfected chordoma cells in vitro. In vivo murine xenograft models of human CH22 and U-CH1 chordoma treated with Delta-24-RGD resulted in tumor volume reduction and enhanced overall survival. RNA-Seq analysis followed by Ingenuity Pathway Analysis predicted activation of tumor-associated extracellular matrix remodeling and host immune response pathways. Analyzing a human chordoma tissue microarray, the immunosuppressive macrophage marker CD163 was associated with shortened recurrence-free survival. Using macrophage/chordoma co-culture, OV infection reduced CD163 expression, induced a concomitant pro-inflammatory macrophage polarization, and enhanced cytotoxicity against chordoma cells. This synergistic cytotoxic effect was further validated in co-cultures of chordoma cell lines with healthy donor and chordoma patient-derived peripheral blood mononuclear cells (PBMCs). Lastly, histological evaluation of patient-derived chordoma tumor slice cultures confirmed infectivity of Delta-24-RGD, underscoring the clinical potential of this oncolytic virus. These studies provide a framework for future clinical translation amongst patients with recurrent or metastatic chordoma.

Glioblastomas (GBM) are the most common and aggressive primary adult brain tumors, and advances in immunotherapy have failed to significantly improve patient outcomes. The presence of the blood brain barrier (BBB), abundance of immunosuppressive myeloid cells, and relative paucity of cytotoxic lymphocytes are major challenges preventing immunotherapy success in GBM. We previously identified the small calcium-binding protein S100A4 as a major regulator of GBM immune suppression. Targeting S100a4 via genetic depletion in tumor or stromal cells is sufficient to reprogram the tumor immune-landscape and extend survival of GBM-bearing mice. We recently developed an S100A4 blocking monoclonal antibody to target S100A4 signaling in the tumor microenvironment; however, this antibody has limited BBB penetrance. To overcome this challenge, we generated a bispecific S100A4-Transferrin-Receptor (TFR) antibody (BsA) that utilizes TFR-mediated transcytosis to cross the BBB. We assessed efficacy of BsA treatment in the preclinical syngeneic S100ß-vErbB;p53 (SVP) GBM mouse model. Using ELISA, confocal immunofluorescent microscopy, and bulk RNA-sequencing, we report that the BsA treatment accumulates in the CNS at nearly twenty-fold the concentration of the monoclonal antibody and reprograms the tumor immune microenvironment to promote immune cell infiltration and pro-inflammatory activation. BsA monotherapy modestly extends survival in the SVP model in an immune-cell dependent manner, as treatment in SVP-tumor bearing NSG mice abolishes this survival benefit. We further assessed if BsA treatment sensitizes immune cold GBMs to immune checkpoint blockade. Combination with αPD-1 checkpoint-blockade nearly doubled median overall survival in the SVP mouse model, with increased tumor infiltrating reactive CD4 and cytotoxic CD8 T cells. Single cell RNA-sequencing revealed an enhanced interferon-γ response signature in GBM cells and macrophages from combination therapy treated-tumors, with an accompanying upregulation of MHC-I and MHC-II associated genes, respectively. These results indicate that the BsA is a promising agent to reprogram the GBM immune-landscape and reactivate anti-tumor immunity.

GBM (glioblastoma) is the most common and aggressive primary brain tumor in adults. Unfortunately, despite aggressive treatments, the median survival is 15.4 months, with less than 5% of patients surviving > 5 year. Therefore, there is urgent need to develop more effective treatments for GBM patients. In theory, immunotherapies are ideal for treating GBM since immune cells can cross the blood brain barrier (BBB), track infiltrating glioma cells, and selectively kill cancer cells while sparing normal brain cells. Unfortunately, most GBM immunotherapy trials, including vaccines, adoptive cellular therapy, CAR-T cells, and immune checkpoint blockade, have provided only modest benefit to GBM patients thus far. A significant barrier that limits efficacy of immunotherapy in GBM and other cancers is the paucity of tumor infiltrating lymphocytes and abundance of immune suppressive myeloid cells. A critical step in enhancing the efficacy of immunotherapy for GBM requires approaches that enhance T cell infiltration and reprogram the immune landscape to more pro-inflammatory state. We recently defined the molecular heterogeneity of glioma and immune cells in human GBM at the single cell level by analyzing >210,000 cells from 44 samples. A major observation from this analysis was that S100A4 is a critical regulator of immune suppressive myeloid and T cells in GBM. Deleting S100a4 in immune cells only was sufficient to reprogram the tumor micronenvironment and significantly extend survival of mice bearing tumors from multiple glioma models. In other cancers, high levels of S100A4 expression is predictive of worse survival and increased metastasis, indicating that S100A4 is a promising immunotherapy target. Based on this and other unpublished results from the lab, we developed a blocking antibody against human S100A4 protein. While we successfully developed antibodies that blocked breast cancer metastases to the lung, our lead antibody did not cross the BBB efficiently to affect glioma growth. To overcome this well-established challenge, we generated a BBB-penetrant bispecific S100A4-TfR antibody that accumulates at therapeutic doses in gliomas. Preliminary results suggest that bispecific antibody (BsA) treatment alone significant extends survival and is associated with increased T cell infiltration and immune remodeling. When combined with anti-PD1 treatment, the median survival nearly doubles in the combination treated mice, compared to IgG control. We are currently performing single cell RNA-sequencing to elucidate molecular and cellular changes induced by the BsA treatment in different cell types and how it synergizes with anti-PD1 treatment in gliomas. Citation Format: Thomas Wong, Jia-Shiun Leu, Xuejun Fan, Reece Kang, Nourhan Abdelfattah, Fransisca Leonard, Ningyan Zhang, Kyuson Yun. A novel bispecific antibody reprograms the immune suppressive TME and sensitize GBM for immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A092.

Treating glioblastoma remains a therapeutic challenge in oncology. Viroimmunotherapy is emerging as a promising new treatment approach. Our laboratory developed an oncolytic adenovirus, termed Delta-24-RGD, which has been tested in clinical trials for recurrent glioblastoma patients with encouraging results (NCT00805376, NCT02798406). These clinical studies, together with preclinical data, showed that the efficacy of Delta-24-RGD was due to direct tumor cell oncolysis and indirect activation of anti-tumor immunity. To further leverage this immune component, our group generated Delta-24-RGDOX, an armed version of Delta-24-RGD that expresses the T-cell activator OX40 ligand (OX40-L). Here, we aim to further improve the efficacy of Delta-24-RGDOX by targeting factors that maintain the immunosuppressive characteristic of gliomas. RNA sequencing of glioma-bearing mice treated with Delta-24-RGDOX revealed interferon gamma (IFNγ) as the top upstream regulator. In addition, we observed upregulation of the tryptophan metabolism and immunoregulator IDO1-AhR network in these treated tumors. We performed gene expression analysis on 38 tumor biopsies from the Delta-24-RGD and Pembrolizumab clinical trial (NCT02798406), identifying a significant positive correlation between IFNγ and IDO1 (a main regulator of AhR activation), identifying this axis as clinically relevant. The IFNγ signaling converges through the JAK family, which induces the activation STATs and the subsequent engagement of inflammatory programs. In vitro studies demonstrated that Delta-24-RGDOX treatment of glioma cells increased STATs and IDO1 expression, resulting in AhR nuclear translocation and transcriptional activation. Of clinical interest, the IFNγ-induced IDO1 expression was completely abrogated by concomitant treatment with Ruxolitinib, a clinically approved JAK inhibitor. Using syngeneic glioma models, we showed that combination therapy with Delta-24-RGDOX and Ruxolitinib yielded better survival outcomes compared to monotherapy controls. In summary, this study illustrates the feasibility of combining virotherapy with drugs targeting immunosuppressive pathways in gliomas and provides a strong rationale for translating this strategy into the clinical setting.

Recent clinical trials have demonstrated that oncolytic virotherapy using adenoviruses can extend survival in a subset of brain tumor patients by promoting T-cell infiltration into otherwise immunologically “cold” tumors (NCT00805376, NCT03178032, and NCT02798406). However, the early detection and elimination of the virus by the host immune system likely limit the therapeutic efficacy of this approach. To address this challenge, we investigated the molecular pathways and sensors responsible for recognizing adenoviruses during infection, with the ultimate goal of modulating this process to improve viroimunotherapy clinical efficacy. RNA-seq analyses revealed the upregulation of Non-Pou Domain-Containing Octamer-Binding (NONO) in intracranial glioma models treated with the oncolytic adenovirus Delta-24-RGD. We demonstrate that NONO is essential for initiating the cGAS-STING pathway and modulating innate immune responses against adenovirus in normal cells and glioma models. Immunoprecipitation and mass spectrometry studies indicate the formation of complexes between NONO and adenovirus DNA-binding protein (DBP), which facilitate cGAS activation. Given that high-grade brain tumors and corresponding cell models often harbor homozygous deletions of downstream interferon effector genes, we further examined the role of the immunotransmitter 2’3’-cyclic GMP-AMP (cGAMP) in local immune activation. Our results show that tumor-derived cGAMP is sufficient to activate local immune cells and that NONO is required for its production following virotherapy. In vivo experiments reveal that downregulation of NONO enhances viral persistence and significantly prolongs overall survival in athymic mice treated with Delta-24-RGD. Collectively, our results identified NONO as a novel sensor of adenoviral DNA and capsid proteins, which is essential for the secretion of cGAMP and activation of innate immune cells. In addition, this data suggests that targeting the NONO-related pathway may guide the development of the next generation of oncolytic viruses to improve the clinical results obtained using viroimmunotherapy and other immunotherapy modalities.